RESUMEN
Group B Streptococcus (Streptococcus agalactiae; GBS) is a leading cause of neonatal sepsis worldwide. As a pathobiont of the intestinal tract, it is capable of translocating across barriers leading to invasive disease. Neonatal susceptibility to invasive disease stems from immature intestinal barriers. GBS intestinal colonization induces major transcriptomic changes in the intestinal epithelium related to barrier function. Butyrate, a microbial metabolite produced by fermentation of dietary fiber, bolsters intestinal barrier function against enteric pathogens, and these effects can be transferred in utero via the placenta to the developing fetus. Our aim was to determine if butyrate mitigates GBS disruption of intestinal barriers. We used human intestinal epithelial cell (IEC) lines to evaluate the impact of butyrate on GBS-induced cell death and GBS adhesion and invasion. IECs and human fetal tissue-derived enteroids were used to evaluate monolayer permeability. We evaluated the impact of maternal butyrate treatment (mButyrate) using our established mouse model of neonatal GBS intestinal colonization and late-onset sepsis. We found that butyrate reduces GBS-induced cell death, GBS invasion, monolayer permeability, and translocation in vitro. In mice, mButyrate decreases GBS intestinal burden in offspring. Our results demonstrate the importance of bacterial metabolites, such as butyrate, in their potential to bolster epithelial barrier function and mitigate neonatal sepsis risk.IMPORTANCEGroup B Streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. It is a commensal of the intestines that can translocate across barriers leading to sepsis in vulnerable newborns. With the rise in antibiotic-resistant strains and no licensed vaccine, there is an urgent need for preventative strategies. Butyrate, a short-chain fatty acid metabolized in the gut, enhances barrier function against pathogens. Importantly, butyrate is transferred in utero, conferring these benefits to infants. Here, we demonstrate that butyrate reduces GBS colonization and epithelial invasion. These effects were not microbiome-driven, suggesting butyrate directly impacts epithelial barrier function. Our results highlight the potential impact of maternal dietary metabolites, like butyrate, as a strategy to mitigate neonatal sepsis risk.
RESUMEN
Mitochondrial dynamic process is important for cell viability, metabolic activity, and mitochondria health. Here, we present a protocol for measuring mitochondrial size through immunofluorescence staining, confocal imaging, and analysis in ImageJ. We describe the steps for tissue processing, antigen retrieval, mitochondrial staining using an integrating immunofluorescence assay, and computerized image analysis to measure each mitochondrial size in mouse and human liver tissues. This protocol reduces tissue sample volume and processing time for the preparation of primary cells. For complete details on the use and execution of this protocol, please refer to Pearah et al.1.